Transcriptome Sequencing Analysis And The Construction Of Binary Vector For ZafA Gene Genetic Transformation Of Trichophyton Mentagrophytes

Trichophyton mentagrophytes is an important zoonotic dermatophytic(ringworm)pathogen that can cause severe skin infections in humans and animals,but the commonly used fungal skin disease prevention and control measures is relatively simple,the study of the Trichophyton mentagrophytes is mostly in the epidemiology and drug efficacy research level,the current study has been unable to meet the needs of clinical medicine.Zinc is an important micronutrient element,which is often binding with a variety of functional proteins to form zinc finger protein to complete a variety of biological functions in fungi and other micro-organisms.At present,a variety of fungi zinc absorption and transport control factor has been clear,such as: Aspergillus fumigatus ZafA protein,Saccharomyces cerevisiae Zap1 protein,as a zinc response activity factor,it can regulate the expression of multiple zinc transporters at the transcription level,thus controlling the ingestion of fungi to zinc ions,playing an important role in the growth and pathogenesis of fungi.However,at present,the composition and function of the zinc-responsive activating factor of Trichophyton mentagrophytes have not been determined.The zinc absorption and transport regulation network is unclear,It is important to determine the zinc-responsive activating factor and the composition of zinc transporters for studing the pathogenicity in Trichophyton mentagrophytes.It was found that the genetic composition of zinc-responsive activating factor of various fungi was similar,thus at the beginning of this experiment,primers were designed based on the sequence of Aspergillus fumigatus zinc-responsive activating factor,PCR was performed on the whole genome DNA and cDNA of Trichophyton mentagrophytes.After several experiments,it was found that the composition of the zinc-responsive activating factor of Trichophyton mentagrophytes was not obtained.Therefore,in this study,the transcriptome of T.mentagrophytes was sequenced using Illumina HiSeqTM 2000 technology under the normal state and the conditions of zinc deficiency,and the de novo assembly of the transcriptome was performed,we also made functional annotation,gene differential expression analysis and so on.The genes related to zinc uptake were preliminarily determined by the transcriptional sequencing data of T.mentagrophytes,and a binary vector was constructed for the transformation of ZafA gene,which laid the foundation for the subsequent verification of gene function.The results are as follows:1.RNA-seq was performed using Illumina sequencing,which generated a total of 10751 Unigenes and more than 89 % of them were annotated,and provided valuable data support for gene function research and assembly of genomic map of T.mentagrophytes.2.The genes related to zinc-uptake were determined by sequence homology comparison with ZafA and Zap1 genes,and the ZafA gene was amplified by high fidelity in T.mentagrophytes.3.ZafA gene flanking fragment and the hygromycin resistance gene(hph)from plasmid pAN7-1 were simultaneously inserted into the binary vector pDHt/SK to construct ZafA gene knockout binary vector pDHt / ZafAⅠ,Ⅱ :: hph.The binary vectors were constructed successfully verified by PCR and restriction enzyme cutting.In summery,the results showed that the transcriptional sequencing and functional annotation of T.mentagrophytes were completed in this study.The composition of ZafA gene of T.mentagrophytes was determined.The binary vector pDHt / ZafAⅠ,Ⅱ :: hph was successfully constructed,which laid the foundation for the subsequent Zaf A gene virulence test and functional validation.